Project Summary Unnatural amino acids, by virtue of unique chemical or physical properties not found in the standard twenty amino acids, can bestow novel properties to the proteins into which they are incorporated. Thus, technologies that enable their incorporation have been developed, and unnatural amino acids have found widespread use in the development of novel protein therapeutics. However, existing methods of unnatural amino acid incorporation have limitations that arise from the fact that they all repurpose the natural genetic code. We hypothesize that orthogonal expansion of the genetic code through the introduction of a third, unnatural base pair (UBP), and subsequent use of this third base pair to incorporate unnatural amino acids into proteins, will circumvent these limitations. The development of a semi-synthetic organism (SSO) capable of replicating, transcribing, and translating UBPs to incorporate unnatural amino acids into proteins is the central goal of this proposal. In specific aim 1, mechanisms that enforce UBP retention will be examined. Specific aim 2 will explore the UBP-mediated incorporation of NCAAs using orthogonal tRNA-synthetase pairs in E. coli. Lastly, specific aim 3 will examine the translational efficiency and fidelity of all 96 possible new codons containing a single UBP. Successful completion of these aims will advance our understanding of the fundamental processes that underlie information storage and retrieval in living organisms. Furthermore, the proposed work has the potential to revolutionize the field of protein therapeutics by providing a robust platform for the incorporation of one or more unnatural amino acids into proteins.